Terpenes are found in most organisms (microorganisms, animals and plants). These compounds are made up of five carbon units called isoprene units and are classified by the number of these units present in their structure. Thus monoterpenes, sesquiterpenes and diterpenes are terpenes containing 10, 15 and 20 carbon atoms respectively. Sesquiterpenes, for example, are widely found in the plant kingdom. Many sesquiterpene molecules are known for their flavor and fragrance properties and their cosmetic, medicinal and antimicrobial effects. Over 300 sesquiterpene hydrocarbons and 3000 sesquiterpenoids have been identified (Joulain, D., and König, W. A., The Atlas of Spectral Data of Sesquiterpene Hydrocarbons, EB Verlag, Hamburg, 1998; Connolly, J. D., Hill R. A., Dictionary of Terpenoids, Vol 1, Chapman and Hall (publisher), 1991), and many new structures are identified each year. Plant extracts obtained by different means such as steam distillation or solvent extraction are used as source of terpenes. Terpene molecules are often used as such, but in some cases chemical reactions are used to transform the terpenes into other high value molecules.
Biosynthetic production of terpenes involves enzymes called terpene synthases. There is virtually an infinity of sesquiterpene synthases present in the plant kingdom, all using the same substrate (farnesyl pyrophosphate, FPP) but having different product profiles. Genes and cDNAs encoding sesquiterpene synthases have been cloned and the corresponding recombinant enzymes characterized. The biosynthesis of terpenes in plants and other organisms has been extensively studied and is not further detailed in here, but reference is made to Dewick, Nat. Prod. Rep., 2002, 19, 181-222, which reviews the state of the art of terpene biosynthetic pathways.
β-Santalene is a naturally occurring sesquiterpene molecule that can be used as starting material for the chemical synthesis or the biosynthesis of β-santalol (as represented in FIG. 2B), which is a major constituent of sandalwood oil. Sandalwood oil is an important perfumery ingredient obtained by distillation of the heartwood of Santalum species. Sandalwood is also largely used for incenses and traditional medicine. The oil contains 90% of sesquiterpene alcohols. Among the different isomers of santalol, β-santalol is the principal contributor to the typical sweet-woody and balsamic odour of sandalwood oil. Other constituents such as epi-β-santalol and α-santalol may also contribute to the sandalwood note.
Generally, the price and availability of plant natural extracts are dependent on the abundance, oil yield and geographical origin of the plants. In addition, the availability and quality of natural extracts is very much dependent on climate and other local conditions leading to variability from year to year, rendering the use of such ingredients in high quality perfumery very difficult or even impossible some years. Due to over-exploitation of the natural resources, difficulties of cultivation, slow growth of the Santalum plants, the availabilities of sandalwood raw material has dramatically decreased during the past decades. Therefore, it would be an advantage to provide a source of β-santalol, which is less subjected to fluctuations in availability and quality. A chemical synthesis of the sandalwood sesquiterpene constituents is so far not available. A biochemical pathway leading to the synthesis of β-santalene, which could then be used to produce β-santalol, would therefore be of great interest.
Santalane type sesquiterpene, and particularly sesquiterpenes with the β-santalane skeleton, were identified in several plant species. Though, no sesquiterpene synthase capable of producing β-santalene, has yet been described.
A sesquiterpene synthase capable of synthesizing at least one bi-cyclic and/or tri-cyclic sesquiterpene having a santalane carbon skeleton, the corresponding nucleic acid and a method for producing such compound having a santalane carbon skeleton are disclosed in the International patent application WO 2006/134523. Nevertheless, no trace of β-santalene was detected as product of the sesquiterpene synthases disclosed in the examples. The only product with a santalane skeleton was epi-beta-santalene. The properties of epi-beta-santalene are very different from those of β-santalene. In particular, it is of no interest in the synthesis of β-santalol. Moreover, the sesquiterpene synthase disclosed in WO 2006/134523 shares only 27% of identity with the sequence of the invention.
The percentage of identity between sesquiterpene synthases known from the databases and the polypeptides of the invention is very low. The closest protein sequence to the β-santalene synthase of the invention is a monoterpene synthase from Santalum album (access No. ACF24767; Jones, C. G., Keeling, C. I., Ghisalberti, E. L., Barbour, E. L., Plummer, J. A. and Bohlmann, J. Arch. Biochem. Biophys., 2008, 477(1), 121-130) which shares 58% amino acid sequence identity with the β-santalene synthase of the invention. When contacted with FPP, this enzyme produces over 90% of β-bisabolene and no santalene isomer is formed.
In addition to the difference between the sequences themselves, it also has to be pointed out that the structure and the properties of the products synthesized by the above-mentioned enzyme are very different from those of the sesquiterpene β-santalene. In particular the monoterpenes produced by this enzyme, i.e. alpha-terpineol, limonene, geraniol, myrcene, linalool and some other minor products are not suitable as a starting material for the synthesis of β-santalol, which is a very useful ingredient in the field of perfumery.
Despite extensive studies of terpene cyclization, the isolation and characterization of the terpene synthases is still difficult, particularly in plants, due to their low abundance, their often transient expression patterns, and the complexity of purifying them from the mixtures of resins and phenolic compounds in tissues where they are expressed.
It is an objective of the present invention to provide methods for making β-santalene in an economic way, as indicated above. Accordingly, the present invention has the objective to produce β-santalene while having little waste, a more energy and resource efficient process and while reducing dependency on fossil fuels. It is a further objective to provide enzymes capable of synthesizing β-santalene, which is useful as perfumery and/or aroma ingredients.